Polyamine conjugated oligonucleotides

ABSTRACT

Oligonucleotide analogs are provided having improved cellular uptake and improved nuclease resistance. Modification of oligonucleotides through the attachment of nitrogenous moieties, especially polyamines, hydrazines and the like to nucleosidic portions of the analogs is disclosed. Oligonucleotides targeted at the tat region of HIV comprise certain preferred embodiments.

FILED OF THE INVENTION

This invention relates to the field of therapeutics, and in particularto the treatment of infection by antisense therapy. This invention alsorelates to the field of gene expression. Novel polyamine conjugatedphosphorothioate oligonucleotides which are useful in antisense therapyare provided. These oligonucleotides have enhanced cellular uptake andconsequently enhanced biological and therapeutic activity. The inventionalso provides methods of synthesis of novel polyamine conjugatedphosphorothioate oligonucleotides.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals includingmost disease states, are effected by proteins. Such proteins, eitheracting directly or through their enzymatic functions, contribute inmajor proportion to many diseases in animals and man. Classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease causing or disease potentiatingfunctions. Recently, however, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, intracellular RNA. These interactions involvedthe binding of complementary "antisense" oligonucleotides or theiranalogs to the transcellular RNA in a sequence specific fashion such asby Watson-Crick base pairing interactions.

The pharmacological activity of antisense oligonucleotides, as well asother therapeutics, depends on a number of factors that influence theeffective concentration of these agents at specific intracellulartargets. One important factor is the stability of the oligonucleotide inthe presence of nucleases. Another key factor is the ability ofantisense compounds to traverse the plasma membrane of specific cellsinvolved in the disease process.

Cellular membranes consist of lipid protein bilayers that are freelypermeable to small, nonionic, lipophilic compounds and inherentlyimpermeable to most natural metabolites and therapeutic agents. Wilson,D. B. Ann. Rev. Biochem. 47:933-965 (1978). The biological antiviraleffects of natural and modified oligonucleotides in cultured mammaliancells have been well documented, so it appears that these agents canpenetrate membranes to reach their intracellular targets. Uptake ofantisense compounds into a variety of mammalian cells, including HL-60,Syrian Hamster fibroblast, U937, L929, CV-1, and ATH8 cells has beenstudied using natural oligonucleotides and nuclease resistant analogs,such as alkyl triesters, Miller, P. S., Braiterman, L. T. and Ts'O, P.O. P., Biochemistry 16:1988-1996 (1977); methylphosphonates,Marcus-Sekura, C. H., Woerner, A. M., Shinozuka, K. Zon, G., andQuinman, G. V., Nuc. Acids Res. 15:5749-5763 (1987) and Miller, P. S.,McParland, K. B., Hayerman, K. and Ts'O, P. O. P., Biochemistry20:1874-1880 (1981); and phosphorothioates, Ceruzzi, M. and Draper, K.Nucleosides & Nucleotides 8:815-818 (1989 ); Miller, P. S., Braiterman,L. T. and Ts'O, P. O. P. Biochemistry 16:1988-1996 (1977) and Loke, S.L., Stein, C., Zhang, X. H. Avigan, M., Cohen, J. and Neckers, L. M.Curr. Top. Microbiol. Immunol. 141:282-289 (1988).

Phophorothioates are oligonucleotide analogs in which the oxygen atom ineach phosphate linkage is replaced by a sulfur. Although the overallcharge is conserved, and they are therefore comparable in that respectto phosphodiester oligonucleotides, several properties of this class ofanalogs makes them more attractive than other modified compounds. Theseinclude ease of chemical synthesis, good aqueous solubility, relativelyhigh resistance to nucleases, and the ability to form stable duplexeswith complementary DNA or RNA strands. However, phosphorothioates werestudied concurrently with natural compounds by Loke et al, Proc. Natl.Acad. Sci. U.S.A. 86:3473-3478 (1989), and while they may be useful dueto their nuclease resistance, they are less efficiently internalizedthan their natural oligonucleotide counterparts.

Advances in nucleotide chemistry have allowed attachment of functionalgroups to the 3' and 5' end of the oligonucleotides to enhance cellularuptake in specific cell types. Previous studies have shown that plasmidDNA complexed with an asiaglycoprotein-poly(L-lysine) conjugate, couldbe targeted to hepatocytes, which contain unique cell surface receptorsfor galactose-terminal (asialo)glycoproteins. Wu, G. Y. and Wu, C. H.Biochemistry 27:887-892 (1988). Other groups have synthesizedoligodeoxyribonucleotides that have a 5'-attached alkylating agent and a3' attached cholesterol moiety and determined that these modifiedoligonucleotides were taken up into cells more efficiently than controlcompounds without the steroid moiety. Zon, G. in Oligodeoxynucleotides:Antisense Inhibitors of Gene Expression 234-247, ed. J. S. Cohen (CRCPress, Boca Raton FL, 1989). Letsinger, et al. Proc. Natl. Acad. Sci.U.S.A. 86:653-656 (1989), have also synthesized cholesteryl-conjugatedphosphorothioates whose anti-HIV activity is significantly greater thannatural oligonucleotides with the same sequence. Additionalmodifications include conjugation of oligonucleotides to poly(L-lysine)alone. Stevenson, M. and Iversen, P. L. J. Gen. Virol 70:2673-2682 (1989) and Lemaitre, M., Baynard, B. and LeBleu, B. Proc. Natl. Acad. Sci.U.S.A. 84:648-652 (1987). This modification enhanced the antiviralactivity of the compound studied presumably due to increased cellularuptake imparted by the polycationic poly(L-lysine).

The activity of antisense oligonucleotides previously available has notbeen sufficient for practical therapeutic, research or diagnostic use.The basis of this insufficiency is likely several fold i.e., (1)incomplete understanding of the secondary and tertiary structure of thetargeted RNA, (2) low percentages of delivery and uptake, (3)inactivation of reactive centers by other cellular components, and (4)requirements for stoichiometric conditions for inhibition of proteinproduction.

Enhancement of cellular uptake of antisense oligonucleotides by chemicalmodification would have clear advantages. Novel modifications may alsolead to increased lipophilicity, greater retention, and increaseddistribution of the novel compounds. Increasing the concentration ofoligonucleotides at specific intracellular target sites may ultimatelyincrease the safety and efficacy of these compounds since less of thedrug will be required to produce the desired effects.

Accordingly, there has been and continues to be a long-felt need foroligonucleotides and oligonucleotide analogs which are capable ofeffective therapeutic and diagnostic antisense use and specifically anoligonucleotide or oligonucleotide analog which is comprised of afunctional group which facilitates transport into the cell and at thesame time is less susceptible to nuclease activity than wild types. Thislongfelt need has not been satisfied by prior work in the field ofantisense oligonucleotide therapy and diagnostics.

SUMMARY OF THE INVENTION

In accordance with this invention, novel types of antisenseoligonucleotides are provided which are modified to enhance celluaruptake. Oligonucleotides having at least one nucleoside modified bydirect attachment to a polyamine have been found to be effective inaccomplishing this goal. It is preferred that the nucleoside be attachedat its 5' site of the sugar moiety to the polyamine. It is preferredthat the oligonucleotides in accordance with this invention be modifiedso as to be phosphorothioates or other backbone modified species. Theoligonucleotides may preferably range in length from about 5 to about 50nucleic acid bases. In accordance with preferred embodiments of thisinvention, the oligonucleotides code for the tat region of a HIV genome.In accordance with other preferred embodiments, the selected sequencecoding for the tat region of the HIV genome has a thymidine at its 5'terminal end. Other preferred antisense oligonucleotide sequencesinclude complementary sequences for herpes, papilloma and other viruses.

The modified nucleoside preferably found at the 5' end of thephosphorothioate oligonucleotide may be any pyrimidine or purine.However, preferred embodiments of this invention incorporate a modifiedthymidine at the 5' end.

The modified nucleosides are preferably nuclease resistant linkages of apolyamine functional group joined to the nucleoside at the 5' site ofits sugar moiety. The polyamine functional group may comprise primaryamines, hydrazines, semi-carbazines, thiosemi-carbazines or similarnitrogenase species. A preferred configuration of the polyaminefunctional group incorporates a symmetrical carbon spacing group betweeneach amine function.

The linkage between the modified nucleoside and the polyamine functionalgroup is preferably, generally unlike functional group additionspresently known in the art. Rather than the usual phosphodiesterlinkage, the addition occurs directly at the 5' position. Not only isthis linkage an improvement over the present state of the art because itis nuclease resistant, but in addition the direct attachment of thisfunctional group to the oligonucleotide or oligonucleotide analogconfers superior cellular uptake relative to the naturally occurringoligonucleotide. This superior cellular uptake may likely be due to theneutralizing effect that the polyamine has on the negative charges ofthe oligonucleotide since the polyamine may be directed back along thesugar-phosphate backbone of the oligonucleotide conjugate as well as thebackbone of the polyamine-oligonucleotide heteroduplex.

This invention is also directed to methods for synthesizing sucholigonucleotides such as routes comprising the synthesis of anintermediate product which may be activated to react with appropriatefunctional groups such as those of the above-mentioned, preferredembodiments. These methods employ the use of solid supports upon whichactivation takes place. Such use of the solid support may either be viaa DNA synthesizer, by manual manipulation of the solid support orotherwise.

OBJECTS OF THE INVENTION

It is a principal object of the invention to provide oligonucleotideanalogs for use in antisense oligonucleotide diagnostics, researchreagents, and therapeutics.

It is a further object of the invention to provide nuclease resistantoligonucleotide analogs which possess enhanced cellular uptake.

Another object of the invention is to provide such oligonucleotideanalogs which are therapeutically safer and which have greater efficacythan naturally-occurring antisense oligonucleotides.

It is yet another object of the invention to provide methods forsynthesis of the modified oligonucleotide using solid supports.

These and other objects will become apparent to persons of ordinaryskill in the art from a review of the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart of certain synthetic schemes useful for thesynthesis of some polyamine functional groups useful in the practice ofthis invention.

FIG. 2 sets forth one method for the synthesis of polyamine conjugatedphophorothioate oligonucleotides useful in this invention.

DETAILED DESCRIPTION OF THE INVENTION

The biological activity of the antisense oligonucleotides previouslyavailable has not generally been sufficient for practical therapeutic,research or diagnostic use. This invention directs itself to modified,naturally-occurring oligonucleotides and analogs and methods foreffecting such modifications. These modified oligonucleotides andoligonucleotide analogs, exhibit increased biological activity relativeto their naturally-occurring counterparts. Furthermore, thesemodifications may be effected using solid supports which may be manuallymanipulated or used in conjunction with a DNA synthesizer usingmethodology commonly known to those skilled in the art.

In the context of this invention, the term "oligonucleotide" refers to aplurality of joined nucleotide units formed in a specific sequence fromnaturally occurring bases and furanosyl groups joined through a sugargroup by native phosphodiester bonds. These nucleotide units may benucleic acid bases such as guanine, adenine, cytosine, thymine oruracil. The sugar group may be a deoxyribose or ribose. This term refersto both naturally occurring or synthetic species formed from naturallyoccurring subunits.

"Oligonucleotide analog" as the term is used in connection with thisinvention, refers to moieties which function similarly tooligonucleotides but which have non naturally occurring portions.Oligonucleotide analogs may have altered sugar moieties or inter-sugarlinkages, for example, phosphorothioates and other sulfur containingspecies which are known for use in the art. Oligonucleotide analogs mayalso comprise altered base units or other modifications consistent withthe spirit of this invention, in order to facilitate antisensetherapeutic, diagnostic or research reagent use of a particularoligonucleotide.

In accordance with the invention, an oligonucleotide sequence isgenerally chosen which is complementary to DNA or RNA which is involvedin the production of proteins whose synthesis is ultimately to bemodulated or inhibited in entirety. One preferred embodiment of thisinvention is an antisense oligonucleotide analog complementary to theDNA or RNA which codes for the tat region of HIV. Other preferredembodiments are directed to herpes and papilloma viruses and stillothers will be apparent to persons of ordinary skill in the art.

Also in accordance with preferred embodiments of this invention,phosphorothioate bonds are substituted for the phosphodiester bondswhich normally comprise the sugar phosphate backbone ofoligonucleotides. These oligonucleotide analogs are preferably furthermodified at their terminal 5' end by the addition of a nucleosideanalog. This nucleoside analog is most preferably a modified thymidine.Said modified nucleoside is so modified by the addition of a protectinggroup such as at the 5' site of the sugar moiety of the thymidine. Theprotecting group in accordance with one preferred embodiment of thisinvention is a 1,3 diphenylimidazolynyl group.

The modified nucleoside analog, having been incorporated into theoligonucleotide analog, is preferably further modified by thereplacement of the protecting group at the 5' site of its sugar moietywith a nitrogenous functional group. The nitrogenous functional group ispreferably a polyamine such as any of primary polyamines, amines,hydrazines, semicarbazides, or thiosemicarbazides but which preferablyis a polyamine having the structures of polyamine 8 or polyamine 10 asset forth in FIG. 1. The intermediate Schiff base or imine orsemicarbazones are reduced to the substituted amine or hydrazine. Mostpreferable the nitrogenous functional group has the structure ofpolyamine 10 as set forth in FIG. 1.

Accordingly, preferred polyamines have the general formula:

    H.sub.2 N(CH.sub.2).sub.n NH(CH.sub.2).sub.n NH(CH.sub.2).sub.n NH--

wherein n is an integer between 2 and 6. It is preferred that n be 4.

In accordance with this invention, polyamines, which are positivelycharged, are caused to lie along the negatively charged sugar-phosphatebackbone of the oligonucleotide. This configuration is believed toneutralize the negative charges of the oligonucleotide and enhance thecellular penetration of the oligonucleotide. Several recent reports havesuggested that a polyamine may form hydrogen bonds to the edges of thebase pairs in the major groove. Loke, S. L., Stein, C. A., Zhang, X. H.,Mori, K., Nakanishi, M., Subashinghe, G., Cohen, J. S. and Neckers, L.M. Proc. Natl. Acad. Sci. U.S.A. 86:3473-3479 (1989). To avoid thisproblem, this invention preferably does not employ the usualphosphodiester conjugation linkage but instead attaches the polyaminedirectly to the 5' position. By eliminating the phosphodiester bond asthe conjugation linker and attaching the polyamine directly to the 5'position, there is believed to be a strong tendency to direct thepolyamine chain back along the sugar phosphate backbone of theoligonucleotide conjugate as well as along the backbone of thepolyamine-oligonucleotide heteroduplex.

Preferred embodiments of this invention employ a polyamine containingabout four carbon units between each amine. Carbon spacers may bearranged in a variety of configurations in relation to the amine groups.This arrangement may be useful in directing the site residence of thepolyamine on the oligonucleotide and its heteroduplex. This ability todirect a polyamine to reside at certain positions of the attachedoligonucleotides and their heteroduplexes will likely have an importantbearing on oligonucleotide uptake and other oligonucleotide properties.Other carbon unit numbers may be included among the nitrogen atoms ofthe preferred polyamines in accordance with this invention. Persons ofordinary skill in the art will have wide latitude in selecting optimumconfigurations for particular circumstances, two, five, six or othernumbers of carbon units may be employed.

In accordance with certain aspects of the invention, FIG. 2 sets forthone chemical synthesis of modified phosphoramidite oligonucleotideanalogs. In particular, FIG. 2 sets forth novel processes involving thesynthesis of 1,3 diphenylimidazolidine protected 5'aldehydic-3'-(2-cyanoethyl N,N diisopropylphosphoramidityl) thymidine.This base-stable thymidine analog is placed at the 5' end of a desiredoligonucleotide sequence and is activated as needed to couple polyaminessuch as primary amines, hydrazines, semi-carbazides, orthiosemicarbazides.

The starting material employed by the method set forth in FIG. 2 isdeoxyribofuranosyl and ribofuranosylnucleosides which can be prepared inhigh yields by the procedure of, or obvious modification of theprocedure of, Pfitzner and Moffatt where the oxidation of 3'-acetylthymidine is described. Pfitzner, K. E. et al., J. American Chem. Soc.85: 3027 (1963). The acetyl derivative of the nucleoside to be modifiedmay also be a suitable starting material and may be commerciallyavailable or may be prepared by a three step process using proceduresknown to those skilled in the art.

The first step of this process is the addition of t-butyldimethylsilylchloride (TBDMSCl) to the selected nucleoside. Second, Ac₂ O is added,followed by the addition of Bu₄ F to produce the final acetyl derivativeof a selected nucleoside. In accordance with the preferred embodiment ofthis invention, the starting material, thymidine, is converted to3'-acetyl thymidine, using the three step process, or alternatively,3'-acetyl thymidine is commercially available.

The 3'-acetyl nucleoside (14) is subsequently oxidized by treatment withDMSO/DCC followed by treatment with 1,2 dianilinoethane to produce3'-O-acetyl-5'-deoxy-5'-(1,3-diphenylimidazolin-2-yl) nucleoside (16).This protecting group has been found to be stable to basic conditionsand can be hydrolyzed back to an aldehyde with mild acid. Any of theexisting or yet to be discovered groups useful in accomplishing thisfunction may be employed in accordance with the practice of the presentinvention. In accordance with preferred embodiments the protectedspecies resulting from the treatment of the 3'-acetyl thymidine withDMSO/DCC followed by treatment with 1,2 dianilinoethane was3'-O-acetyl-5'-deoxy-5'-(1,3-diphenylimidazolin-2-yl) thymidine. Theprotected nucleoside is next transformed to a phosphoramidite (18) bystandard procedures of base deprotection through the addition of NH₃/MeOH and subsequent phosphitylation by the addition of phosphityl Cl.The preferred method being phosphitylation by the addition of2-cyanoethyl N,N diisopropylchlorophosphoramidite to producediphenylimidazolinylthymine phosphoramidite.

Separately, a phosphorothioate oligonucleotide having a preselectedsequence is extended on a solid support in a 5' direction until suchpoint at which a nucleoside corresponding to the specific modifiednucleoside prepared above need be incorporated into the oligonucleotidesequence. The modified nucleoside is substituted for its naturallyoccurring terminal nucleoside counterpart (20). Most preferably amodified thymidine replaces its naturally occurring counterpart at the5' end. Synthesis may be carried out conveniently through solid statesynthesis employing known phosphoramidite methodology on a DNAsynthesizer or otherwise. Nucleic acid synthesizers are commerciallyavailable and their use is generally understood by persons of ordinaryskill in the art as being effective in generating nearly anyoligonucleotide of reasonable length which may be desired. Sucholigonucleotide chain may be from about 5 to about 50 nucleic acid basesin length. It is more preferred that such functionalities have from 8 toabout 40 base units and even more preferred that from about 12 to 20base units be employed. At present, it is believed that oligonucleotideanalogs having about 15 base units will likely be found to be best forthe practice of certain embodiments of the present invention.

According to a preferred embodiment of this invention, the tat region ofHIV is extended in the 5' direction to produce the sequenceCCGCCCCTCGCCTCTTGCCT because this region is believed to have the mostpotent activity in inhibiting the expression of the tat protein. SeeU.S. Pat. Ser. No. 521,907, incorporated by reference, filed May 11,1990 and assigned to the Assignee of this invention. The sequence isthen preferably extended three additional nucleotides toTCGCCGCCCCTCGCCTCTTGCCT in order to allow for a T nucleotide on the 5'end. The thymine residue was substituted with adiphenylimidazolinylthymidine as described above.

The resulting phosphoramidite linkage may be preferably sulfurized usingthe known Beaucage reagent, Beaucage, S. L. et al., J. Am. Chem. Soc.112:1253-1254 (1990), to afford preferred phosphorothioates, or byiodine to afford the phosphodiester linkage.

The addition of a nitrogenous functional group, preferably a polyaminesuch as those incorporating primary amines, hydrazines, semicarbazides,or thiosemicarbazides, may be preferably effected once the modifiednucleoside has been added to the phophoramidite oligonucleotide. Thefirst step in this addition is the deprotection of the modifiednucleoside which has been added to the 5' end of the oligonucleotide.This deprotection can occur, according to preferred embodiments bytreatment of the control pore glass (CPG) support (which binds thephosphoramidite oligonucleotide in the synthesizer) with 3%dichloroacetic acid (DCA) in THF. Thereafter, the CPG support is treatedwith a polyamine so as to form a Schiff's base which is reduced to anamine by the addition of sodium cyanoborohydride (NaCHBH₃). Finally thecolumn is treated with ammonium hydroxide to deprotect and remove thepolyamine-oligonucleotide conjugate (22) from the support. Treatment ofthe CPG support may be effected by a DNA synthesizer or manually usingsyringes. The preferred functional groups for the embodiment of thisinvention are polyamines. Most preferably, the polyamine functionalgroup useful in combination with other preferred components of thisinvention, is tris(aminobutyl)amine. This polyamine was chosen becauseof preferred length. The length of these amine-carbon linkagesapproximately spans the length of a 15 base pair oligonucleotide basedupon molecular modeling. Carbon spacers placed between each amine may beuseful in directing the polyamine to reside at certain positions of theattached oligonucleotide. Preferably, carbon spacers will be arrangedsuch that the carbon spacing group will be symmetrical. Four carbonspacers placed between each amine is a preferred embodiment of thisinvention.

Synthesis of the polyamine group may be performed by procedures known inthe art. For preferred embodiments, the synthesis of the polyamine groupcan be performed according to the steps set forth in FIG. 1. Thesynthesis of tris(aminobutyl)amines (7) can be obtained by proceduresdescribed by Niitsu and Samejima, Chem. Pharm. Bull. 34(3):1032-1038(1986), incorporated by reference herein. Such amines will be protectedsuch as with a t-butyldiphenylsilyl group at one of the primary aminesaccording to the procedure set forth by Miller and Braiterman, et al.,Biochemistry 16:1988-1996 (1977), which involves the addition ofTNDPS-Cl and Et₃ N. The resulting t-butyl diphenyl silyamine (8) is aconvenient amine for reaction with the modified oligonucleotide. Morepreferably, however, this compound will be further modified bytrifluoroacetylation with trifluoroethyl acetate in Et₂ N to producepolyamine 9 (9) followed by selective removal of thet-butyldiphenylsilyl moiety with pyridinium hydrogen fluoride to providea protected amine (10). This deprotection process is described byOverman, et al, Tetrahedron Lett. 27:4391-4394 (1986). The overallprocess, is amendable to the synthesis of a wide variety of polyamine inhigh overall yield. All mentioned references are incorporated byreference herein.

In the alternative, the 5' aldehyde of a nucleoside may be condensedwith a trifluoroacetyl protected polyamine. Subsequent reduction of theSchiff's base with sodium cyanoborohydride and phosphitylation of the 3'position will afford a protected polyamine monomer. This protectedpolyamine monomer may be attached to the 5' end of the oligonucleotidevia the DNA synthesizer.

Deprotection and removal of the polyamineoligonucleotide conjugate fromthe column may be performed by treatment of the column with ammoniumhydroxide. Finally, the composition may be purified using HPLC and gelelectrophoresis systems. Such purification procedures are well known bythose skilled in the art.

The following examples are illustrative, but not limiting, of theinvention.

EXAMPLE 1 Preparation of 1,3 Diphenylimidazolynyl

3' acetyl thymidine, prepared through standard procedures known in theart, was treated with DMSO/DCC. This treatment was followed by treatmentof the mixture with dianilinoethane to produce 3'-O-acetyl 5' deoxy-5'(1,3-diphenylimidazolin-2-yl)thymidine according to procedures ofPfitzner and Moffatt.

2. Phosphoramidite Production

3'-O-acetyl 5' deoxy 5' (1,3 diphenylimidazolin-2-yl)thymidine wastransformed to the phosphoramidite using standard procedures of basedeprotection and followed by phosphitylation to produce 2-cyanoethylN,N-diisopropylchlorophosphoramidite.

3. Preparation of Oligonucleotide Sequence

Extend the active HIV phosphorothioate oligonucleotide with the sequence5' CCGCCCCTCGCCTCTTGCCT 3' (464A) in the 5' direction until a thymidineresidue was encountered. The addition of three additional nucleotides toproduce an oligonucleotide of the sequence 5' TCGCCGCCCCTCGCCTCTTGCCT 3'was performed by standard automated synthesis on an ABI model 380B DNAsynthesizer. The terminal thymidine was then replaced bydiphenylimidazolinylthymidine.

4. Preparation of Polyamine

Prepared tris (aminobutyl)amine for addition to the oligonucleotide bystandard procedures known to those skilled in the art.

5. Addition of Polyamine

The phosphoramidite linkage was sulfurized with Beaucage reagent.Beaucage, S. L. et al., J. Am. Chem. Soc. 112: 1253-1254 (1990). Next,the CPG support was treated with 3% dichloroacetic acid in THF. The CPGsupport was subsequently treated withtris(trifluoroacetyl)tris(aminobutyl)amine to form Schiff's base. Sodiumcyanoborohydride (NaCNBH₃) was added to reduce the conjugate to anamine. The column was treated with ammonium hydroxide to deprotect andremove the conjugate from column support. The polyamine conjugate waspurified with ion exchange HPLC, using a Beckman Gold HPLC system, andwith gel electrophoresis.

EXAMPLE 2

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of papilloma virus mRNA cap region, 5'TATGCAAGTACAAAT 3', is performed.

EXAMPLE 3

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of papilloma virus mRNA cap region, 5'TATGCAAGTACAAAT 3', is performed.

EXAMPLE 4

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of papilloma virus initiation oftranslation sequence, 5' TCTCCATCCTCTTCACT 3', is performed.

EXAMPLE 5

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of herpes virus, 5' TCATCCATCCTTCGGCC3', is performed.

EXAMPLE 6

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of herpes virus 5' TGGCCATTTCAACAGA 3',is performed.

EXAMPLE 7

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of herpes virus 5' TCATCCATCCGTCCGCC 3',is performed.

EXAMPLE 8

Using the protocol set forth in Example 1, the synthesis of an antisensephosphoramidite oligonucleotide of herpes virus 5' TTGGCCATTGGAACCAA 3',is performed.

What is claimed is:
 1. An oligonucleotide analog having at least onenucleoside modified by direct attachment of a polyamine at the 5' siteof its sugar moiety.
 2. The oligonucleotide analog of claim 1 whereinthe oligonucleotide analog ranges in length from about 5 to about 50nucleotide base unit.
 3. The oligonucleotide analog of claim 1 whereinsaid nucleoside is thymidine.
 4. The oligonucleotide analog of claim 1wherein said polyamine is a primary amine, a hydrazine, a semicarbazide,or a thiosemicarbazide.
 5. The oligonucleotide analog of claim 1specifically hybridizable with the tat region of an HIV genome.
 6. Theoligonucleotide analog of claim 5 wherein said selected sequence has athymidine at its 5' terminal end.
 7. The oligonucleotide analog of claim1 specifically hybridizable with a selected portion of a herpes genome.8. The oligonucleotide of claim 1 specifically hybridizable with aselected portion of a papilloma genome.
 9. the oligonucleotide analog ofclaim 1 wherein said polyamine has the structure:

    H.sub.2 N(CH.sub.2).sub.n NH(CH.sub.2).sub.n NH(CH.sub.2).sub.n NH--

wherein n is an integer between 2 and
 6. 10. The oligonucleotide analogof claim 9 wherein n is
 4. 11. The oligonucleotide analog of claim 1comprising at least one phosphorothioate group.